The nature of this Mentored Clinical Scientist Development Award (K08) pertains to the understanding of human pluripotent and hematopoietic stem cells. This proposed 5-year research training program will advance the study of human developmental hematopoiesis with one of the most important new tools in biomedical research: the human embryonic stem cell (hES). My long-term interest is in advancement of clinical stem cell transplantation, and I intend to devote a large part of my career to basic research in stem cell biology. This proposal will focus on the derivation, culture, and expansion of primitive human hematopoietic stem cells (HSC) from hES. The strategy for identifying early human HSC will rely on the general hypothesis that a "hemangioblast" (bipotential progenitor of HSC and endothelium) and/or a "hemogenic endothelial" precursor can be physically isolated and characterized from differentiating hES using experimental approaches similar to those described in other species. These hES-derived progenitors will be used for subsequent studies in human lympho-hematopoietic development. The research program will initially focus on expanding, purifying, and characterizing hES-derived hemangioblasts. Subsequent studies will probe the mechanistic role SCL/TAL1, a master regulator for initiating hematopoiesis, plays on the formation of the human hemangioblast. A reporter cDNA will be gene-targeted to the endogenous SCL locus regulatory region ("knock-in") with modified methods of homologous recombination, and primitive SCL-expressing hemato-endothelial progenitors will be purified and expanded for further phenotypic and genetic analysis. Primitive HSC will ultimately be cultured on stromal lines and/or fetal thymic organ cultures for the derivation of a model for human T-lymphocyte development. The role of Notch pathway signaling in hES-drived lymph-hematopoietic tissue will be studied as a long-term goal. The success of these goals will lay the groundwork for characterizing human stem cells not available by any other means.